UNASSIGNED: Natural cases of prion disease have not been reported in rabbits, and prior attempts to identify a prion conversion agent have been unsuccessful. However, recent applications of prion seed amplifying experimental techniques have sparked renewed interest in the potential susceptibility of rabbits to prion disease infections. Among several factors related to prion disease, polymorphisms within the prion-like protein gene (PRND), a member of the prion protein family, have been reported as significantly associated with disease susceptibility in various species. Therefore, our study aimed to investigate polymorphisms in the PRND gene of rabbits and analyze their genetic characteristics.
UNASSIGNED: Genomic DNA was extracted from 207 rabbit samples to investigate leporine PRND polymorphisms. Subsequently, amplicon sequencing targeting the coding region of the leporine PRND gene was conducted. Additionally, linkage disequilibrium (LD) analysis was employed to assess the connection within and between loci. The impact of non-synonymous single nucleotide polymorphisms (SNPs) on the Doppel protein was evaluated using PolyPhen-2.
UNASSIGNED: We found nine novel SNPs in the leporine PRND gene: c.18A > G, c.76G > C, c.128C > T, c.146C > T, c.315A > G, c.488G > A, c.525G > C, c.544G > A, and c.579A > G. Notably, seven of these PRND SNPs, excluding c.525G > C and c.579A > G, exhibited strong LD values exceeding 0.3. In addition, LD analysis confirmed a robust link between PRNP SNP c.234C > T and PRND SNPs at c.525G > C and c.579A > G. Furthermore, according to PolyPhen-2 and SIFT analyses, the four non-synonymous SNPs were predicted to have deleterious effects on the function or structure of the Doppel protein. However, PANTHER and Missense3D did not indicate such effects.
UNASSIGNED: In this paper, we have identified novel SNPs in the rabbit PRND gene and predicted their potential detrimental effects on protein function or structure through four non-synonymous SNPs. Additionally, we observed a genetic linkage between SNPs in the PRND and PRNP genes. These findings may provide insights into understanding the characteristics of rabbits as partially resistant species. To the best of our knowledge, this study is the first to genetically characterize PRND SNPs in rabbits.

Di-n-butyl phthalate (DBP), a well-known endocrine disruptor, causes male reproductive dysfunction. To understand the underlying mechanisms, we performed histological, endocrinological, and biochemical analyses and assessed the expression of genes involved in spermatogenesis and sperm function according to OECD test guideline 407. Following 28 days of administration of the lowest observed adverse effect level dose of DBP to mice, no significant changes in body weight, testis and epididymis weights and histology, serum testosterone level, or testicular daily sperm production were found. Nonetheless, the motility of the epididymal sperm of the DBP group was significantly decreased together with an increase in the incidence of bent tails and abnormal heads. In the testes of the DBP group, lipid peroxidation (LPO) level was significantly increased and testicular Bcl-2 mRNA level was significantly decreased together with an increase in the Bax/Bcl-2 mRNA ratio. In the testes of the DBP group, levels of Prnd mRNA and protein and Pou4f1 mRNA, an activator of the Prnd promotor, were significantly decreased. Of note, prion-like protein doppel (PRND) was significantly decreased together with decreased PRND immunoreactivity in the head, midpiece, and tail of sperm. In the testes of the DBP group, levels of Sox9, Sgp1, and Sgp2 mRNA, which are functional Sertoli cell markers, were significantly decreased. Level of Amh mRNA, a Sertoli cell immaturity marker, was significantly increased together with that of Inha mRNA, suggesting deregulation of the brain-gonadal axis. Together, our findings suggest that DBP at present dosage may potentiate LPO generation and Sertoli cell immaturity via downregulation of Sox9 and disruption of the Pou4f1-Prnd gene network in post-meiotic germ cells without visible changes in spermatogenesis or testosterone level. This may result in structural and functional abnormalities in spermatozoa. Additionally, our findings suggest that assessment of the male reproductive toxicity of phthalate ester plasticizers based on conventional OECD test guidelines should be reconsidered.

Prion disease is a fatal infectious disease caused by the accumulation of pathogenic prion protein (PrPSc) in several mammals. However, to date, prion disease has not been reported in horses. The Sho protein encoded by the shadow of the prion protein gene (SPRN) plays an essential role in the pathomechanism of prion diseases. To date, the only genetic study of the equine SPRN gene has been reported in the inbred horse, Thoroughbred horse. We first discovered four SPRN single nucleotide polymorphisms (SNPs) in 141 Jeju and 88 Halla horses by direct DNA sequencing. In addition, we found that the genotype, allele and haplotype frequencies of these SNPs of Jeju horses were significantly different from those of Halla and Thoroughbred horses, this latter breed is also included in this study. Furthermore, we observed that the minimum free energy and mRNA secondary structure were significantly different according to haplotypes of equine SPRN polymorphisms by the RNAsnp program. Finally, we compared the SNPs in the coding sequence (CDS) of the SPRN gene between horses and prion disease-susceptible species. Notably, prion disease-susceptible animals had polymorphisms that cause amino acid changes in the open reading frame (ORF) of the SPRN gene, while these polymorphisms were not found in horses.

BACKGROUND: Chronic wasting disease (CWD) is a cervid prion disease that is caused by abnormal prion protein (PrPSc ). Recent studies have reported that prion family genes showed a strong association with the susceptibility of several types of prion diseases. To date, an association study of the prion-related protein gene (PRNT) has not been performed in any type of cervid prion disease.
METHODS: In the present study, we investigated PRNT polymorphisms in large deer, including 235 elk, 257 red deer and 150 sika deer. We compared genotype, allele and haplotype frequencies of PRNT polymorphisms between CWD-negative animals and CWD-positive animals to find an association of PRNT polymorphisms with the susceptibility of CWD.
RESULTS: We found a total of five novel single nucleotide polymorphisms (SNPs) in the cervid PRNT gene. Interestingly, we observed significantly different distributions of genotypes and allele frequencies of three PRNT SNPs, including c.108C>T, c.159+30C>T and c.159+32A>C, between CWD-negative and CWD-positive red deer. In addition, significant differences of two haplotype frequencies in red deer were found between the CWD-negative and CWD-positive groups. However, the association identified in the red deer was not found in elk and sika deer.
CONCLUSIONS: To the best of our knowledge, this report is the first to describe the strong association of PRNT SNPs with the susceptibility of CWD.

Prion disease is a fatal neurodegenerative disorder caused by a deleterious prion protein (PrPSc). However, prion disease has not been reported in horses during outbreaks of transmissible spongiform encephalopathies (TSEs) in various animals in the UK. In previous studies, single nucleotide polymorphisms (SNPs) in the prion protein gene (PRNP) have been significantly associated with susceptibility to prion disease, and strong linkage disequilibrium (LD) between PRNP and prion-like protein gene (PRND) SNPs has been identified in prion disease-susceptible species. On the other hand, weak LD values have been reported in dogs, a prion disease-resistant species. In this study, we investigated SNPs in the PRND gene and measured the LD values between the PRNP and PRND SNPs and the impact of a nonsynonymous SNP found in the horse PRND gene. To identify SNPs in the PRND gene, we performed direct sequencing of the PRND gene. In addition, to assess whether the weak LD value between the PRNP and PRND SNPs is a characteristic of prion disease-resistant animals, we measured the LD value between the PRNP and PRND SNPs using D\' and r2 values. Furthermore, we evaluated the impact of a nonsynonymous SNP in the Doppel protein with PolyPhen-2, PROVEAN, and PANTHER. We observed two novel SNPs, c.331G > A (A111T) and c.411G > C. The genotype and allele frequencies of the c.331G > A (A111T) and c.411G > C SNPs were significantly different between Jeju, Halla, and Thoroughbred horses. In addition, we found a total of three haplotypes: GG, AG, and GC. The GG haplotype was the most frequently observed in Jeju and Halla horses. Furthermore, the impact of A111T on the Doppel protein was predicted to be benign by PolyPhen-2, PROVEAN, and PANTHER. Interestingly, a weak LD value between the PRNP and PRND SNPs was found in the horse, a prion disease-resistant animal. To the best of our knowledge, these results suggest that a weak LD value could be one feature of prion disease-resistant animals.

Prion disease has displayed large infection host ranges among several species; however, dogs have not been reported to be infected and are considered prion disease-resistant animals. Case-controlled studies in several species, including humans and cattle, indicated a potent association of prion protein gene (PRNP) polymorphisms in the progression of prion disease. Thus, because of the proximal location and similar structure of the PRNP gene among the prion gene family, the prion-like protein gene (PRND) was noted as a novel candidate gene that contributes to prion disease susceptibility. Several case-controlled studies have confirmed the relationship of the PRND gene with prion disease vulnerability, and strong genetic linkage disequilibrium blocks were identified in prion-susceptible species between the PRNP and PRND genes. However, to date, polymorphisms of the dog PRND gene have not been reported, and the genetic linkage between the PRNP and PRND genes has not been examined thus far. Here, we first investigated dog PRND polymorphisms in 207 dog DNA samples using direct DNA sequencing. A total of four novel single nucleotide polymorphisms (SNPs), including one nonsynonymous SNP (c.149G>A, R50H), were identified in this study. We also found two major haplotypes among the four novel SNPs. In addition, we compared the genotype and allele frequencies of the c.149G>A (R50H) SNP and found significantly different distributions among eight dog breeds. Furthermore, we annotated the c.149G>A (R50H) SNP of the dog PRND gene using in silico tools, PolyPhen-2, PROVEAN, and PANTHER. Finally, we examined linkage disequilibrium between the PRNP and PRND genes in dogs. Interestingly, we did not find a strong genetic linkage between these two genes. To the best of our knowledge, this was the first genetic study of the PRND gene in a prion disease-resistant animal, a dog.

Prion protein (PrP(C)) biosynthesis involves a multi-step process that includes translation and post-translational modifications. While PrP has been widely investigated, for the homolog Doppel (Dpl), limited knowledge is available. In this study, we focused on a vital step of eukaryotic protein biosynthesis: targeting by the signal recognition particle (SRP). Taking the ovine Dpl (OvDpl(1-30)) peptide as a template, we studied its behavior in two different hydrophobic environments using CD and NMR spectroscopy. In both trifluoroethanol (TFE) and dihexanoyl-sn-glycero-3-phosphatidylcholine (DHPC), the OvDpl(1-30) peptide revealed to fold in an alpha-helical conformation with a well-defined central region extending from residue Cys8 until Ser22. The NMR structure was subsequently included in a computational docking complex with the conserved M-domain of SRP54 protein (SRP54M), and further compared with the N-terminal structures of mouse Dpl and bovine PrP(C) proteins. This allowed the determination of (i) common predicted N-terminal/SRP54M polar contacts (Asp331, Gln335, Glu365 and Lys432) and (ii) different N-C orientations between prion and Dpl peptides at the SRP54M hydrophobic groove, that are in agreement with each peptide electrostatic potential. Together, these findings provide new insights into the biosynthesis of prion-like proteins. Besides they also show the role of protein conformational switches in signalization toward the endoplasmic membrane, a key event of major significance in the cell cycle. They are thus of general applicability to the study of the biological function of prion-like as well as other proteins.